Experimental Monitoring and Modeling of Oxygen Dynamics in Laccase-Catalyzed Phenolic Oxidation

Abstract

Oxygen-dependent enzymes such as laccases play a central role in green oxidation processes, yet kinetic analyses often overlook the dynamic role of dissolved oxygen. In this study, we propose a useful methodology for the experimental monitoring and kinetic modeling of oxygen-consuming enzymatic reactions, demonstrated using the laccase-catalyzed oxidation of gallic acid and ferulic acid. The method integrates real-time monitoring of dissolved oxygen and substrate concentration with dynamic simulations accounting for oxygen mass transfer and bi-substrate kinetic models commonly used to describe laccase-catalyzed reactions. Experiments were conducted in both non-sparged and air-sparged systems to assess kinetic behavior under oxygen-limited and oxygen-sufficient conditions. The method yielded well-constrained kinetic constants for gallic acid and revealed a biphasic oxygen-uptake pattern for ferulic acid, arising from secondary oxidation of radical intermediates. Using Aspen Custom Modeler built-in non-linear regression and numerical integration algorithms, kinetic parameters were estimated by fitting them to complete time-course data, enabling accurate modeling of complex oxygen-dependent reactions. The proposed framework provides a practical tool for studying oxidase-catalyzed processes under conditions where dissolved-oxygen dynamics can be experimentally resolved, and supports more rational design of biocatalytic systems where oxygen availability governs the activity, selectivity and control of complex reaction networks.